A research program will be undertaken in the area of TGF-p signaling, a fundamental cellular process that is implicated in multiple human diseases, including cancer. A key feature of the signaling pathway is phosphorylation of a family of latent transcription factors termed R-Smads, a biochemical event that leads to nuclear accumulation. The central hypothesis of this research is that continuous nucleocytoplasmic shuttling of R-Smads with repeated cycles of receptor-mediated phosphorylation and nuclear dephosphorylation, permits constant sensing of the activation status of the receptor and hence efficient termination of signaling upon receptor inactivation. To explore this, we will study the three critical phases in the biochemical lifetime of the R-Smad, Smad2, namely, bis-phosphorylation of the protein by the activated TpR-l receptor, nuclear import of the phosphorylated Smad2/Smad4 complex, and nuclear dephosphorylation of Smad2 followed by its nuclear export. Key to this research program is our ability to introduce biochemical and biophysical probes site-specifically into phosphorylated forms of T[3R-I and Smad2, thereby allowing us to control and monitor their activities. Chemistry-driven protein engineering approaches will be used in conjunction with established biophysical and cell biological approaches to study the detailed mechanisms by which Smad2 interacts with the activated receptor complex, and thereafter shuttles to and from the nucleus. The specific aims are: 1. To Study the Mechanisms Underlying R-Smad Activation: We will use biochemical and structural techniques to investigate how activated TpR-l receptor recognizes and then double phosphorylates Smad2. 2. To Study the Mechanisms Underlying R-Smad Nuclear Import: We will use biochemical and cell biological techniques to study the molecular mechanisms by which phosphorylation of Smad2 leads to its nuclear accumulation. 3. To Study the Mechanisms Underlying R-Smad Nuclear Export. We will identify and characterize the putative nuclear phosphatase responsible for removing the activation phosphates from Smad2. By providing a more complete quantitative and structural understanding of the basic signaling pathway, this research program will lay a firmer foundation for the rational design of small molecular therapies based on manipulation of TGF-psignaling.